The complete chloroplast genome of Gardenia stenophylla Merr (Rubiaceae) and its phylogenetic analysis

Abstract Gardenia stenophylla Merr, a member of the genus Gardenia in the family Rubiaceae, possesses significant medicinal and ornamental value and is widely distributed in China. This study reports the newly sequenced chloroplast genome of Gardenia stenophylla Merr. The complete chloroplast genome of Gardenia stenophylla Merr (155,109 bp, GC content of 37.5%) was shown to have a typical quadripartite structure, containing a pair of inverted repeat regions (IRs) of 28,802 bp separated by a large single-copy (LSC) region of 85,396 bp and a small single-copy (SSC) region of 18,109 bp. The chloroplast genome contained 151 genes encoding 106 proteins, 37 tRNAs, and eight rRNAs. The Gardenia stenophylla Merr chloroplast genome displayed the closest phylogenetic relationship to Gardenia jasminoides and Gardenia jasminoides var. grandiflora. These data will assist in future molecular phylogenetics of the Rubiaceae.


Background
About 250 species of Gardenia are distributed in tropical and subtropical regions of the Eastern Hemisphere.There are only six species of Gardenia naturally distributed in China, of which only Gardenia jasminoides is widely distributed in provinces south of the Yangtze River, while the natural distribution of the other species is extremely restricted (Li et al. 2017).Gardenia stenophylla Merr., 1922 is only distributed in a few areas of Guangdong, Guangxi, Hainan, and other provinces.Its plants are considered graceful with fragrant and beautiful flowers, so it is often planted as an ornamental.However, its fruits and roots are used for medicinal purposes, having cooling and detoxifying effects (Zheng et al. 2012).Notably, G. jasminoides is a well-known medicinal plant both domestically and internationally, while G. stenophylla is also used as a source of the traditional Chinese medicine Gardeniae Fructus in some provinces such as Guangdong.G. stenophylla displays narrow-lanceolate or linear-lanceolate leaves, which are distinctly narrower than the G. jasminoides.The fruit of G. stenophylla Merr is oblong with a diameter often less than 1.3 cm, which is also significantly smaller than that of G. jasminoides.When using EST-SSR to analyze the genetic relationships in Gardenia spp., G. stenophylla Merr showed a closer genetic relationship to G. jasminoides than the ornamental varieties of G. jasminoides var.fortuniana (Deng et al. 2021).
Given that G. stenophylla is utilized as a substitute for the traditional Chinese medicine Gardeniae Fructus in certain regions, investigating the rationality of its use as a source plant for this medicinal preparation is of significant importance.Consequently, an in-depth analysis of the phylogenetic position of G. stenophylla within the genus Gardenia and its genetic relationship with G. jasminoides has become particularly crucial.The study on the chloroplast genome sequencing and phylogenetic relationship of G stenophylla Merr not only provides essential reference for the selection of related species in Gardenia breeding but also contributes to the diversification of medicinal, ornamental, and edible plant resources within the genus.Furthermore, this research will provide scientific evidence for the taxonomic classification of Gardenia species.The outcomes of this study are expected to have substantial implications for the molecular systematics of Gardenia, conservation of genetic diversity, and the rational utilization of medicinal plant resources within this genus.

Materials and methods
Gardenia stenophylla Merr was obtained from the Guangxi Province and planted in greenhouse conditions at the Jiangxi Academy of Forestry (Figure 1), Nanchang, China (28.74 � N, 115.82 � E).The voucher specimen was deposited in the Tree herbarium, Jiangxi Academy of Forestry (voucher number: XYZZ01; contact: Shaoyong Deng, jxforestry@163.com).Total genomic DNA of fresh leaves was extracted with TGuide plant genomic DNA prep kit (Tiangen Biotech, Beijing, China).

Results
The total length of the chloroplast genome of Gardenia stenophylla Merr (GenBank accession no.OL517769) was 155,109 bp with an overall GC content of 37.5%.
To validate the accuracy of the assembled genome sequence, we mapped sequencing reads back to the assembled sequence.We achieved a depth coverage ranging from �1388 to �6184 across the assembled genome, with an average depth of �3820.32 (Supplementary Figure 1).The results indicate that the assembly is reliable.
To probe the phylogenetic relationship of G. stenophylla with the family Rubiaceae, a phylogenetic tree was constructed from the chloroplast genomes of G. stenophylla, 16 other Rubiaceae members and that of Gentiana scabra (Gentian family) as an outgroup.The result confirmed that G. stenophylla is a member of Gardenia and indicates that it displays the closest genetic relationship with G. Jasminoides.

Discussion and conclusions
In this study, we assembled and annotated the chloroplast genome sequence of G. stenophylla Merr for the first time.We found that the chloroplast genome structure of G. stenophylla includes a pair of IRs, a SSC, and a LSC, which is consistent with the chloroplast genome structure of most angiosperms.Detailed annotation of the genome revealed protein-coding genes, tRNA genes, and rRNA genes.To determine the phylogenetic position of G. stenophylla within the Rubiaceae, we conducted a ML phylogenetic analysis.The results indicated that G. stenophylla indeed belongs to the genus Gardenia and shares the closest genetic relationship with G. jasminoides.This finding provides important insights into the evolutionary relationships within the genus Gardenia.Additionally, we observed that the chloroplast genome size of G. stenophylla does not significantly differ from those of other published Rubiaceae species (Zhang et al. 2021).
In conclusion, this study reports the complete chloroplast genome sequence of G. stenophylla for the first time, providing valuable foundational data for further studies in the molecular systematics, evolutionary biology, and genetic diversity of the genus Gardenia and the Rubiaceae family.This information serves as a crucial reference for expanding the medicinal plant resources of the genus Gardenia and for parent selection in the hybrid breeding of G. jasminoides.Moreover, it holds significant reference value for future studies in conservation genetics and molecular breeding of Rubiaceae plants.

Figure 1 .
Figure 1.Morphological characteristics of Gardenia stenophylla Merr.(A-D) Photos of leaves, immature fruit, ripe fruit and obverse and reverse views of the leaves, respectively (photos taken by Shaoyong Deng in the greenhouse facility at the Jiangxi Academy of Forestry, Nanchang, China).

Figure 3 .
Figure 3. Circular map of the complete chloroplast genome of Gardenia stenophylla Merr.The map contains six tracks.From the center going outward, the first circle shows the distribution of sequence repeats connected by either red (the forward direction) or green (the reverse direction) arcs.The second circle shows the distribution of long tandem repeats as short blue bars.The third track shows the distribution of microsatellite sequences as short bars with different colors.The fourth circle shows the sizes of the chloroplast genome regions, including the small single-copy (SSC), inverted repeat (IRA and IRB), and large single-copy (LSC) regions.The fifth track shows the GC content along the genome.The sixth and outer circle shows the distribution of genes colored according to their functional group (legend on the left).Genes indicated inside the circle are transcribed clockwise, whereas genes indicated outside the circle are transcribed anticlockwise.